To gain insight into the structural mechanism of the conformational conversion process of prion, we examined the potential amyloidogenic property of each secondary structural element in a mouse prion protein (mPrP) and discriminated their relative significance for the formation of amyloid fibrils. Although peptides corresponding to alpha-helix 2 and alpha-helix 3 (named H2 peptide and H3 peptide, respectively) formed the amyloid-like fibrils, their structures were quite different. H2 fibrils formed the ordered beta-sheet with the beta-turn conformation, and the resultant fibrils were long and straight. In contrast, H3 fibrils consisted of the beta-sheet with the random conformation, and the resultant fibrils were short and flexible. These properties are basically consistent with their hydrophobicity and beta-strand propensity profiles. To examine the cross reactivity between peptide fragments and full-length mPrP, we then carried out seeding experiments. While H2 seeds induced the formation of fibrils of full-length mPrP as quickly as full-length mPrP seeds, H3 seeds exhibited a long lag time. This implies that the region of alpha-helix 2 rather than alpha-helix 3 in mPrP has great potential for initiating fibril formation. As a whole, the alpha-helix 2 region would be crucial for the nucleation-dependent replication process of the prion protein.